Laboratory: Unit 3: Gram stain & microscopy; inoculate broth (pages 52-53) Lecture: PCR & ribosomal RNA-based phylogeny In-Class Writing: describe colonies.

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Laboratory: Unit 3: Gram stain & microscopy; inoculate broth (pages 52-53) Lecture: PCR & ribosomal RNA-based phylogeny In-Class Writing: describe colonies (page 51); peer review lab report 2 ( ) Hand In: lab report 2 draft (pages 37-39) Read: manual (pages 52-53); Day, chapter 37 Due Next Class: lab report 2; flow chart 3

Due Class 9: lab report 2 (pages ) Send your lab report to:

Identification of bacterial species by PCR amplification and sequencing 16S ribosomal RNA genes.

Zone of clearing around colony indicates antibiotic production.

Antibiotic- producing colony of Bacillus subtilis

Polymerase Chain Reaction (PCR) Amplify specific DNA from complex mixture. PCR is: repetitive primer-directed DNA synthesis using heat-stable DNA polymerase to extend primers annealed to opposite strands of template.

double-stranded template DNA 94 o C 50 o C denature anneal primers 72 o CDNA polymerase Cycle 1

94 o C 50 o C denature anneal primers 72 o CDNA polymerase + + Cycle 2 + +

94 o C 50 o C denature anneal primers 72 o CDNA polymerase + Cycle

94 o C 50 o C denature anneal primers 72 o CDNA polymerase Cycle 4

Cycle 5

16S ribosomal RNA (partial sequence) PCR primers: 27F & 519R

Purify PCR product prior to sequencing binding buffer (PB) = high salt wash buffer (PE) = high salt + ethanol

Purify PCR product prior to sequencing 50 ul distilled water dissolve in 50 ul distilled water ready for sequencing